下调miR-183靶向肿瘤抑制因子CPEB1增强脑胶质瘤细胞的放射敏感性

Down-regulation of miR-183 targeted tumor suppressor CPEB1 enhances radiosensitivity of glioma cells

目的:探讨miR-183对脑胶质瘤细胞放射敏感性的影响。方法:2020年10月至2021年6月,收集秦皇岛市第一医院40例脑胶质瘤组织标本,对T98G细胞进行梯度剂量X射线(0、2、4、6 Gy)照射。采用qPCR检测miR-183、细胞质多腺苷酸化元件结合蛋白1(cytoplasmic polyadenylation element-binding protein 1,CPEB1)在脑胶质瘤组织、T98G细胞和经X射线照射的T98G细胞中的表达量。将miR-183 inhibitor转染T98G细胞后下调miR-183表达,经6 Gy X射线垂直照射,CCK-8法、流式细胞术和WB法,分别检测T98G细胞增殖能力、细胞凋亡率及BAX和Bcl2蛋白表达量。Targetscan软件预测和双荧光素酶报告基因实验检测miR-183与CPEB1的靶向关系。下调CPEB1表达后,经6 Gy X射线照射,分别用CCK-8法、流式细胞术和WB法检测T98G细胞增殖能力、细胞凋亡率及BAX和Bcl2蛋白表达量。将pcDNA-CPEB1或CPEB1 siRNA质粒转染T98G细胞,分别下调或过表达CPEB1后,检测miR-183通过CPEB1对T98G细胞放射敏感性的影响。结果:脑胶质瘤组织和细胞中miR-183呈高表达,CPEB1 mRNA呈低表达。T98G细胞中miR-183的表达量随着X射线放射剂量增加而降低(P<0.05),CPEB1表达量随着X射线放射剂量增加而升高(P<0.05)。6 Gy X射线照射T98G细胞后,下调miR-183可降低细胞增殖能力、增加细胞凋亡率,而过表达miR-183则起到相反作用(P<0.05)。miR-183靶向CPEB1 mRNA且负调控CPEB1表达。下调CPEB1表达后,经6 Gy X射线照射可显著提高T98G细胞增殖能力(P<0.05)、降低细胞凋亡率(P<0.05),miR-183可逆转CPEB1过表达对细胞T98G放射敏感性的促进作用(P<0.05)。结论:下调miR-183的表达能够负调控CPEB1,从而增强脑胶质瘤细胞的放射敏感性。

Objective:To investigate the effect of miR-183 on radiosensitivity of glioma cells.Methods:From October 2020 to June2021,tissue samples of 40 cases of brain glioma from the First Hospital of Qinhuangdao were collected.T98 G cells were irradiated with gradient dose X-ray(0,2,4,6 Gy).The expression of miR-183 and CPEB1 in glioma tissues,T98 G cells and X-ray irradiated T98 G cells were detected by qPCR.After transfection of T98 G cells with miR-183 inhibitor,the expression of miR-183 was down-regulated.The proliferation ability,apoptosis rate,BAX and Bcl2 protein expression of T98 G cells were detected by CCK-8 assay,flow cytometry and WB,respectively,after 6 Gy X-ray vertical irradiation.The targeting relationship between miR-183 and CPEB1 was detected by Targetscan software prediction and dual luciferase reporter gene assay.After down regulating the expression of CPEB1,after 6 Gy X-ray irradiation,the proliferation ability,apoptosis rate,BAX and Bcl2 protein expression of T98 G cells were detected by CCK-8 assay,flow cytometry and WB,respectively.T98 G cells were transfected with pcDNA-CPEB1 or CPEB1 siRNA plasmid,and after CPEB1 was down-regulated or overexpressed,respectively,the effect of miR-183 on radiosensitivity of T98 G cells through CPEB1 was detected.Results:The expression of miR-183 in glioma tissues and cells increased significantly,and the expression of CPEB1 mRNA decreased significantly.The expression of miR-183 in T98 G cells decreased with the increase of X-ray radiation dose(P<0.05),and the expression of CPEB1 increased with the increase of X-ray radiation dose(P<0.05).After 6 Gy X-ray irradiation of T98 G cells,down-regulation of miR-183 could reduce cell proliferation and increase apoptosis rate,while overexpression of miR-183 had the opposite effect(P<0.05).MiR-183 targeted mRNA CPEB1 and negatively regulated CPEB1 expression.After down-regulation of CPEB1,6 Gy X-ray irradiation could significantly improve the proliferation of T98 G cells(P<0.05)and reduce the apoptosis rate(P<0.05).MiR-183 could reverse the promoting effect of CPEB1 overexpression on radiosensitivity of T98 G cells(P<0.05).Conclusion:Down-regulation of miR-183 expression can negatively regulate CPEB1,which might enhance the radiosensitivity of glioma cells.